Monte Carlo simulations and phantom modeling for spatial frequency domain imaging of surgical wound monitoring

Postoperative surgical wound infection is a serious problem around the globe, including in countries with advanced healthcare systems, and a method for early detection of infection is urgently required. We explore spatial frequency domain imaging (SFDI) for distinguishing changes in surgical wound healing based on the tissue scattering properties and surgical wound width measurements. A comprehensive numerical method is developed by applying a three-dimensional Monte Carlo simulation to a vertical heterogeneous wound model. The Monte Carlo simulation results are validated using resin phantom imaging experiments. We report on the SFDI lateral resolution with varying reduced scattering value and wound width and discuss the partial volume effect at the sharp vertical boundaries present in a surgical incision. The detection sensitivity of this method is dependent on spatial frequency, wound reduced scattering coefficient, and wound width. We provide guidelines for future SFDI instrument design and explanation for the expected error in SFDI measurements

Early stage dental caries detection using near infrared spatial frequency domain imaging

Early stage dental caries can be remineralized without the need for “drill-and-fill” treatments that are more invasive and less permanent. However, early stage caries lesions typically present as a white spot on a white background, resulting in many lesions only being identified after they have developed beyond the point of remineralization as cavities. We present a spatial frequency domain imaging technique to characterize the optical properties of dental tissue. This technique enables different dental tissue types (healthy enamel, healthy dentin and damaged or demineralized enamel) to be easily distinguished from one another and allows quantification of the reduced scattering coefficients of dental tissue. The use of near-infrared light at 850 nm allows high depth penetration into the tissue and suppression of absorption effects, ensuring only changes in the reduced scattering coefficient that result directly from demineralization of enamel are observed and simplifying the analysis method. This technique provides a tool to both guide the attention of dentists to areas of interest and potential demineralization, and to provide longitudinal quantified assessments to monitor caries lesion behaviour over time

Spectral index selection method for remote moisture sensing under challenging illumination conditions

Remote sensing using passive solar illumination in the Short-Wave Infrared spectrum is exposed to strong intensity variation in the spectral bands due to atmospheric changing conditions and spectral absorption. More robust spectral analysis methods, insensitive to these effects, are increasingly required to improve the accuracy of the data analysis in the field and extend the use of the system to “non ideal” illumination condition. A computational hyperspectral image analysis method (named HIAM) for deriving optimal reflectance indices for use in remote sensing of soil moisture content is detailed and demonstrated. Using histogram analysis of hyperspectral images of wet and dry soil, contrast ratios and wavelength pairings were tested to find a suitable spectral index to recover soil moisture content. Measurements of local soil samples under laboratory and field conditions have been used to demonstrate the robustness of the index to varying lighting conditions, while publicly available databases have been used to test across a selection of soil classes. In both cases, the moisture was recovered with RMS error better than 5%. As the method is independent of material type, this method has the potential to also be applied across a variety of biological and man-made samples

Integrated fiber optic spectrally resolved downwelling irradiance sensor for pushbroom spectrometers

We present an integrated fiber optic spectrally resolved downwelling irradiance sensor for pushbroom hyperspectral imagers. The system comprises of a cosine corrector and custom fiber patch cables, collecting the ambient light in a large solid angle and feeding it directly to the entrance slit of the spectrometer. The system enables simultaneous measurement of downwelling and upwelling irradiance using the main hyperspectral camera sensor. As a demonstration, the spectral reflectance of a soil sample was measured with a RMSE of 8.4%, a significant improvement on the RMSE of 54% found without correction. At a weight of approximately 10 grams, this system provides a substantial weight saving over standalone incident light sensing instruments

Pressure-dependent regulation of Ca2+ signaling in the vascular endothelium

The endothelium is an interconnected network upon which hemodynamic mechanical forces act to control vascular tone and remodeling in disease. Ca2+ signaling is central to the endothelium's mechanotransduction and networked activity. However, challenges in imaging Ca2+ in large numbers of endothelial cells under conditions that preserve the intact physical configuration of pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca2+ signaling. We developed a miniature wide-field, gradient-index (GRIN) optical probe designed to fit inside an intact pressurized artery which permitted Ca2+ signals to be imaged with subcellular resolution in a large number (∼200) of naturally-connected endothelial cells at various pressures. Chemical (acetylcholine) activation triggered spatiotemporally-complex, propagating IP3-mediated Ca2+ waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3-mediated Ca2+ signals in all activated cells. By computationally modeling Ca2+ release, endothelial shape changes were shown to alter the geometry of the Ca2+ diffusive environment near IP3 receptor microdomains to limit IP3-mediated Ca2+ signals as pressure increased. Changes in cell shape produce a geometric, microdomain-regulation of IP3-mediated Ca2+ signaling to explain macroscopic pressure-dependent, endothelial-mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3-mediated Ca2+ signaling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that provides access to hundreds of endothelial cells in intact arteries in physiological configuration

Flicker-assisted localization microscopy reveals altered mitochondrial architecture in hypertension

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic “flickers” of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension

Chapter 9 Mitochondria Structure and Position in the Local Control of Calcium Signals in Smooth Muscle Cells

Features of Ca2+ signals including the amplitude, duration, frequency and location are encoded by various physiological stimuli. These features of the signals are decoded by cells to selectively activate smooth muscle functions that include contraction and proliferation [1–3]. Central, therefore, to an appreciation of how smooth muscle is controlled is an understanding of the regulation of Ca2+

Use of fiber optic technology to measure the effects of anesthesia on luciferase reaction kinetics

In vivo bioluminescent imaging (BLI) is a sensitive and reliable technique for studying gene expression, although experiments must be controlled tightly to obtain reproducible and quantitative measurements. The luciferase reaction depends on the availability of the reaction substrate, oxygen, and ATP, the distribution of which can vary markedly in different tissues. Here we used in vivo fiber optic technology, combined with stereotaxis-assisted surgery, to assess luciferase reaction kinetics in response to 2 anesthetic regimens, isoflurane and ketamine–xylazine. Transgenic rats that expressed luciferase under the control of the human prolactin promoter were used as a model organism. Anesthesia had a marked effect on luciferase reaction kinetics. The rise time to peak emission differed by 20 min between isoflurane and ketamine–xylazine. Optical imaging using a charge-coupled–device camera confirmed this delay. These results demonstrate that different anesthetics can have substantial effects on luciferase reaction kinetics and suggest that the timing of image acquisition after substrate injection should be optimized in regard to experimental conditions and the tissues of interest

Design, Manufacture, and Evaluation of Prototype Telescope Windows for Use as Low Vision Aids

Pixellated Optics, a class of optical devices which preserve phase front continuity only over small sub areas of the device, allow for a range of uses that would not otherwise be possible. One potential use is as Low Vision Aids (LVAs), where they are hoped to combine the function and performance of existing devices with the size and comfort of conventional eyewear. For these devices a Generalised Confocal Lenslet Array (GCLA) is designed to magnify object space, creating the effect of traditional refracting telescope within a thin, planar device. By creating a device that is appreciably thinner than existing LVA telescopes it is hoped that the comfort for the wearer will be increased. We have developed a series of prototype GLCA-based devices to examine their real-world performance, focussing on the resolution, magnification and clarity of image attainable through the devices. It is hoped that these will form the basis for a future LVA devices. This development has required novel manufacturing techniques and a phased development approach centred on maximising performance. Presented here will be an overview of the development so far, alongside the performance of the latest devices

Mitochondria structure and position in the local control of calcium signals in smooth muscle cells

In smooth muscle mitochondria are major regulators of contractility, proliferation and growth through the organelles' control of cytoplasmic Ca2+ concentrations. Mitochondria regulate cytoplasmic Ca2+ over concentrations of the ion that range from 200nM – 50 µM. An acknowledged feature of the organelle’s ability to control Ca2+ over the higher Ca2+ concentrations (>10 µM) is the position and structure of the organelles at sites near ion channels. However, the precise relationship between Ca2+ signalling and mitochondria is preliminary in large part because the structure and position of the organelles is not well understood. We recently developed methods to determine the structure and position of each mitochondrion and the entire organelle complement in live, fully-differentiated cells smooth muscle cells. In fully differentiated smooth muscle, mitochondria are distributed through the cytoplasm mainly as spherical or short rod shaped structures (mean length 0.9 µm). Mitochondrial Ca2+ uptake regulates Ca2+ release from IP3R clusters. However, the organelles do not appear to regulate the gating of voltage-dependent Ca2+ channels on the plasma membrane. Nonetheless the position of mitochondria correlates with an increased magnitude of voltage-dependent Ca2+ entry. Voltage-dependent Ca2+ channel expression or distribution, or both, may be regulated by mitochondria